The present invention relates to a method enabling the immunogenicity of an antigen to be increased, and to the use of products capable of being obtained, in particular as a vaccine.
The human cytomegalovirus (CMV), a herpesvirus in the latent state in immunocompetent individuals, is responsible for a wide variety of pathologies which are often fatal in the case of immunosuppression (foetus, graft, AIDS, leukaemia, cancer) (for review see (1)). CMV is the main source of congenital malformations of infectious origins. For all these reasons, CMV poses a serious public health problem. The battle against this virus is limited to chemotherapy with non-specific antivirals such as Ganciclovir(copyright) and Foscarnet(copyright), the side effects and nephrotoxicity of which have been described. As regards prevention, passive immunotherapy (injection of gamma globulins) decreases the incidence of primary infection in seronegative marrow grafts. In transplant patients (marrow, kidney, liver, heart, liver allografts), CMV infection is associated with graft rejection. The recipients, under immunosuppressant (cyclosporin) are treated with Ganciclovir(copyright) during transplantation, and receive gamma globulins during the three months following surgery. The intravenous injection of immunoglobulins at high dose in these patients has permitted a reduction in the incidence of pneumonia and rejection in some cases. This gives rise to the not insignificant problem of the very high cost associated with these treatments. Opportunistic CMV infection in HIV+ individuals is among the most dramatic, and necessitates chemotherapy with antivirals.
The economies achieved by elaborating a policy of prevention of diseases associated with CMV infection in individuals at risk would be substantial. In effect, estimates of the cost associated with vaccinating an individual and with covering the attendant expenses (serological analyses, vaccine, treatment of minor side effects), carried out in the United States (2), show that this cost appears to be about 50 times lower than that of the care provided for a newborn infant who is a victim of a congenital infection. CMV infection is observed in ⅔ of renal transplant patients and at a high incidence in other transplant patients. If it is associated with complications in about ⅓ of them, the annual cost added to that of transplantation is considerable. Despite the impact on the disease of drugs such as Ganciclovir(copyright) or the injection of gamma globulins, the prevention of the primary infection and of reactivation in these patients should be a priority. The benefits of immunization are obvious from both a clinical and an economic standpoint.
The elaboration of a CMV vaccine should make it possible to curb the development of pathologies associated with congenital infections by vaccinating young women before and during pregnancy, to provide for the protection of patients awaiting transplantation and to initiate an anti-cytomegalovirus response in asymptomatic HIV+ individuals. The use of cured virus does not appear to be suitable, since viral extracts do not in general induce a cytotoxic TCD8+ type response. Attenuated virus gives rise to the problem of the oncogenic character, latency and reactivation of the viral particles. The development of recombinant vaccines which should enable these risks to be avoided necessitates knowledge of the most immunogenic antigens or antigen fragments (epitopes) of the infectious agent. The aim of vaccination is the induction of a protective immunity. Rational approach to vaccination should involve three steps: (i) identification of the effector mechanisms responsible for protection, (ii) choice of an antigen capable of inducing a response in all individuals, and (iii) use of an administration route for the vaccine which is capable of inducing the desired type of response (humoral: antibodies, cellular: cytotoxic and helper).
The body""s defence against a viral infection is brought about essentially by the development of a humoral immune response (production of neutralizing antibodies) preventing adsorption of the virus to the cell surface on the one hand, and a cellular response (cytotoxic TCD8+ cells and TCD4+ helper cells) removing infected cells and inhibiting viral replication (synthesis of cytokines (TNFxcex1, IFN-xcex3, etc.)) on the other hand. As regards human cytomegalovirus, among the 200 proteins encoded by the double-stranded DNA (230 kbp), three of them are the respective major targets of these different types of response: the envelope glycoprotein gB (3), the tegument phosphoprotein pp65 (4) and the regulatory phosphoprotein IE1 (5).
While the elaboration of a CMV vaccine appears to be a necessity, there remains the general problem of the mode of conveying the recombinant antigens. The antigens introduced into synthetic structures must be capable of initiating or restoring a lasting B and T (CD4+ helper and cytotoxic CD8xe2x88x92) immune response, thereby effecting protection of the individuals against a primary infection, a reinfection or a reactivation of the latent virus. The activation of T cells (naive cells or memory cells) is linked to the capacity for presentation of the antigen by the antigen presenting cells, the most important of which are the dendritic cells, B lymphocytes and macrophages. In this context, the vector particles will have to permit access to the endogenous (class I, TCD8+) and exogenous (class II, TCD4+) presentation pathways.
The company Biovector Therapeutics has developed a type of structure called Biovecteur Supramolxc3xa9culaire [Supramolecular Biovector] consisting of a polysaccharide core (PSC) covered with a surrounding outer layer of fatty acids (AC) or of phospholipids (SMBV), the composition of which can be varied. The polysaccharide mesh possesses an adjustable degree of crosslinking, can be functionalized (anionic or cationic radical), is very stable and permits the binding of a large amount of antigen inside the core as well as at its periphery.
Unexpectedly, the Applicant found that the combination of a protein or peptide with these vectors enabled a potentiation of the immunogenic response to be observed, relative to that brought about by administration of the antigen alone.
Accordingly, the subject of the present invention is a method for increasing the immunogenicity of an antigen, characterized in that the antigen is combined via stable interactions with a particulate vector, said vector containing:
a non-liquid hydrophilic core
an outer layer of compounds chosen from the group comprising phospholipids and fatty acids.
Preferably, the core consists of a matrix of naturally or chemically crosslinked polysaccharides or oligosaccharides. According to one of the aspects of the invention, ionic ligands are grafted onto the core, it being possible for this ligand to carry, in particular, a function chosen from the group comprising phosphates, sulphates, carboxylic acids, quaternary ammonium groups, secondary amines and primary amines.
The vectors permitting implementation of the method can be between 10 nm and 5 xcexcm in size, and especially advantageous results are obtained with sizes between approximately 25 nm and 200 nm, in particular of the order of 80 nm.
The combination of an antigen with acylated or phospholipid-containing particulate vectors significantly increases the proliferative response of TCD4+ cell clones in vitro to this antigen, in the presence of isolated peripheral blood leukocytes and B/EBV lymphocytes. The yields of combination of the antigen with the particles are high, and the complexes obtained are very stable in physiological medium. The demonstration of a potentiation of the proliferative T response underlines the exceptional properties of these biovectors and the value of their potential use in vaccination.
These results are not obtained with a simple mixture, without prior interaction, of the antigen and the particulate vector.
The antigen is combined with the particulate vector via ionic and/or hydrophobic bonds.
According to an aspect of the invention, the outer layer of the particulate vector consists of natural fatty acids bound to the core via covalent bonds. According to another aspect, this outer layer consists of natural or synthetic phospholipids.
The subject of the invention is also a product capable of being obtained by the method described, and which comprises a combination potentiating the immunogenicity of an antigen bound via ionic and/or hydrophobic bonds to a particulate vector, the said particulate vector containing
a non-liquid hydrophilic core, and
an outer layer, combined with the core via hydrophobic interactions and/or ionic bonds and consisting of lipids compounds chosen from the group comprising phospholipids and fatty acids.
The antigen can be a protein or peptide of bacterial or viral origin, or a fragment derived from these antigens.
More especially, the invention relates to a product potentiating immunogenicity, as defined above, in which the antigen is a protein of CMV or a fragment of such a protein.
The exceptional properties of these vectors can, in effect, be exploited for the encapsulation of antigens of human CMV, with the aim of designing a recombinant vaccine which would combine the IE1, pp65 and gB antigens for the reasons mentioned above. Such a product would be capable of generating a humoral and cytotoxic response.
The antigen can hence be, in particular, the protein IE1 of CMV, or a fragment of this protein.
A fragment of the recombinant viral protein IE1 of human cytomegalovirus (Towne strain) was produced and purified in the form of a fusion protein (GST-e4) in E. coli. The combination of the protein with different types of particle (PSC, AC and SMBV) 80 nm in size is very stable and the yields are very high. We have described an adjuvant effect of the antigen (Ag)/particle complexes on the proliferative response of specific TCD4+ clones in vitro. This effect was observed using peripheral blood leukocytes and EBV-transformed B lymphocytes (B/EBV) as antigen presenting cells.
The proliferation of anti-IE1 T clones in the presence of recombinant GST-e4, soluble or combined with particles, suggests that coupling of the viral antigen to the bacterial GST, the absence of phosphorylation of the protein in this prokaryotic expression system and the combination with biovectors have had no influence on the nature of the peptide epitopes generated by the presenting cells. One of the hypotheses, by which the Applicant in no way intends to limit the invention, is that the adjuvant effect observed when the antigen is combined with the particles might be correlated with a larger uptake of protein by the presenting cell than when the antigen is soluble, as has been found by the Applicant. The concentration of the antigen in the endolysosomal compartments might favour the interaction of class II molecules with viral peptides rather than with endogenous or exogenous peptides originating from serum proteins, for example. The outcome of this would be the expression of a larger number of DR-peptide IE1 complexes at the cell surface. The potentiating effect observed with ACs might be linked to a better accessibility of the antigen. The vectors we used enabled a large mass of antigen to be incorporated by simple mixing in aqueous solution. This represents a considerable advantage relative to the protocols for encapsulation in other types of particle such as liposomes (6).
It is possible that the observed adjuvant effect might also be mediated by an opsonisation of the particles, involving interaction between serum IgGs or complement fragments such as C3b and their respective receptors at the surface of the presenting cells.
The Applicant has shown that a fusion protein which is more stable than e4 alone, between the fragment e4 of CMV and glutathione S-transferase (GST), prepared by genetic engineering, may be used in the vectors according to the invention without necessitating a cleavage step in order to remove the GST portion. This represents a considerable advance, since such a protein can be readily purified, and the elimination of the cleavage step enables the yield to be increased and its large-scale production to be envisaged.
Hence the subject of the invention is also a fusion protein, characterized in that it comprises the fragment e4 of the protein IE1 of CMV and at least part of the glutathione S-transferase protein.
The aim of vaccination is to induce the maturation of cells involved in the specific removal of a pathogenic agent and to maintain clones of memory cells specific for the target antigens in the circulation and in the secondary lymphoid organs. The important contribution of B cells as antigen presenting cells to the activation of T cells has been reported (7). It has been shown that the B cell is effective in taking up soluble antigens at low concentration by endocytosis only by means of its surface immunoglobulin. Studies designed to target liposomes on B lymphocytes make use of the binding to their surface of monoclonal antibodies (8) designed to interact with the surface immunoglobulins of these cells. Since the incidence of specific B lymphocytes is very low, the effect observed here using particles should enable the specific T response of the complexed antigen to be amplified without the latter undergoing a recognition by specific B lymphocytes. The presentation of an Ag to activated TCD4+ cells induces, besides their proliferation, the secretion of cytokines and the expression of surface molecules which are important for the proliferation and differentiation of B cells to plasma cells secreting antibodies.
According to another aspect, the subject of the invention is a pharmaceutical composition, characterized in that it comprises an antigen combined with a particulate vector according to the specification defined above, dispersed in pharmaceutically acceptable excipients.
Its subject is also a product or a composition as defined above, for their use as a vaccine.